Role And Signalling Of Endoplasmic Reticulum Stress In Endogenuous Cardiomyocyte Of Hypoxic Preconditioning And Hypoxic Postconditioning

Ischemic heart disease, one of the leading causes of death in the world, demands early and complete reperfusion in therapy strategy. However, post-ischemic reperfusion may worsen myocardial damage, and result in irreversible ischemia/reperfusion (I/R) injury. Ischemic preconditioning (IPC), multiple brief ischemic episodes before prolonged ischemia, and postconditioning (I-postC), brief intermittent ischemia during the onset of reperfusion, both are known as endogenous protective mechanism, which could markedly limit myocardial infarction and render the myocardium more resistant to the subsequent I/R injury.Endoplasmic reticulum (ER) is the most important organelle in eukaryocyte, serving for macromolecules biosynthesis, stress action and calcium homeostasis adjusting. Given the importance of this organelle, it is exquisitely sensitive to alterations in homeostasis. Stimuli in the course of I/R, including hypoxia, oxidant stress, ATP depletion, Ca²⁺ overload, etc. can affect ER function, namely ER stress (ERS). ERS induces ER molecular chaperones such as glucose-regulated proteins(GRPs), calreticulin(CRT), folding enzymes, etc., which are protective and benefit for cell adaptation. However, when persistent or too intense, ERS will induce and activate ER proapoptosis fators such as CHOP, caspase-12, and JNK. Depended on the duration and level of stress, cells eventually result in adaptation for survival or apoptosis. Then, is there any correlation between ERS and the protective mechanism of IPC and I-post C? The present study tested the involvment of ERS in cardioprotection of HPC and H-postC in cultured neonatal cardiomyocyte subjected to hypoxia /reoxygenation (H/R).Neonatal cardiomyocytes were prepared from Sprague-Dawley rats aged 24 hours. H/R was induced by 2 hours' hypoxia(H) followed by 14 hours' reoxygenation(R), HPC by H-20 min/ R-24 h before H/R, and H-postC by 3 circles of R-5 min/H-5 min at the beginning of reoxygenation after 2 hours' hypoxia. The inhibitors of p38 MAPK and JNK, SB203580 and SP600125, were added respectively to the medium 5-10 min prior to HPC or H-postC.Morphological studies, lactate dehydrogenase (LDH) leakage, and flow cytometry were employed to assess cell apoptosis and necrosis. The expression of GRP78 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). CRT expression and caspase-12 activation, phosphorylation of p38 MAPK and JNK were detected using Western blot analysis. All experiments were repeated at least four separate times. Variables were analyzed by one-way ANOVA for multiple comparisons, and a P value<0.05 was considered significant.(1) HPC and H-postC protect cardiomyocytes from H/R injury.Both HPC and H-postC relieve cell injury caused by H/R, including increase in cell survive rate, decrease in cell apoptosis rate, and LDH leakage.(2) Effect of HPC and H-postC on ERS molecules' expression/ activation. H/R induced up-regulation of Grp78 mRNA, CRT expression and caspase-12activation. Both HPC and H-postC were found to increase the up-regulation of Grp78 mRNA, and relieve CRT over-expression and caspase-12 activation induced by H/R. But H-postC showed less effect than HPC on inhibiting caspase-12 activation induced by H/R.(3) The signal pathway of HPC and H-postC in mediating ERS.When SB203580 was presented before HPC and H-postC, the protection of HPC and H-postC decreased, and CRT expression was markedly suppressed, and the inhibition effect of HPC on caspase-12 activation was almost eliminated. Pretreating cells with SP600125, the protective effect, and the inhibition on CRT expression and caspase-12 activation of HPC had not been markedly affected; however, the depressive effect of H-postC on caspase-12 activation was enhanced.Conclusion: Both HPC and H-postC induced comparable ERS-related cardioprotection mediated by p38 MAPK. However, HPC was more powerful than H-postC in suppressing caspase-12 activation. JNK activation promoted caspase-12 activation and might contribute to ERS-related cell apoptosis induced by H/R.